The present invention relates to a method of treatment of mast cell mediated inflammatory disorders in mammals.
The occurrence of allergic diseases has increased during the last decades, especially in the western world. Most allergic diseases are caused by IgE-mediated (immunoglobulin E) hypersensitivity reactions. Upon activation, during an allergic reaction, IgE molecules bind, via a cell-binding constant domain to specific receptors (Fcxcex5RI) on mast cells.
Mast cells are generally considered to be long living cells, that are widely distributed throughout vascularized tissues and certain epithelia where they play a fundamental role in the pathogenesis of immediate hypersensitivity reactions. In inflammatory disorders, such as allergies and asthma increased numbers of mast cells in affected tissues have been documented with a positive correlation between mast cell number and the severity of the allergic response symptoms. (Otsuka, H., J. Denburg, J. Dolovich, D. Hitch, P. Lapp, R. S. Rajan, J. Bienenstock, and D. Befus. 1985. Heterogeneity of metachromatic cells in human nose: Significance of mucosal mast cells. J. Allergy Clin. Immunol. 76:695-702.)
Upon activation and Fcxcex5RI aggregation, mast cells release preformed mediators such as histamine, proteases, tryptase and heparin, and synthesize lipid mediators and cytokines causing the allergic response observed in sensitized individuals. Survival, growth and differentiation of mast cells are regulated by cytokines such as interleukin-3 (IL-3) and stem cell factor (SCF). Mast cells have the capacity to survive the activation induced degranulation process, and to subsequently regranulate, enabling them to be activated again. Multiple rounds of mast cell activation may underlie the mechanism regulating the reoccurring inflammatory attacks of allergic patients during a pollen season. The possibility of regulating the longevity of activated mast cells could thus provide a treatment for mast cell mediated inflammatory disorders, such as allergies and asthma. However, the molecular mechanisms enabling activated mast cells to survive have not been elucidated.
A well documented mechanism for regulating cell longevity and survival is through the up-regulation of pro-survival genes belonging to the bcl-2 protein family. This is a growing family of apoptosis-regulatory genes, which may either be death antagonists (bcl-2, bcl-XL, bcl-w, mcl-1 and Al) or death agonists (bax, bak, bcl-XS, bad, bid, bik and hrk) [Reed, J. 1994. Bcl-2 and the regulation of programmed cell death. J. Cell Biol 124:1-6)]. Although the precise mechanism by which bcl-2 family members influence apoptosis is unkown, several lines of evidence suggest that bcl-2 proteins function at a critical decision point immediate upstream of an irreversible commitment to cell death [Dragovich, T., C. M. Rudin, and C. B. Thompson. 1998 Signal dransduction pathways that regulate cell survival and cell death. Oncogene 17:3207-13.]. It has been demonstrated that nerve growth factor (NGF) markedly increases bcl-2 expression in mast cells. When overexpressed, bcl-2 prolongs survival of mast cells following IL-3 deprivation. SCF and IL-3 induce suppression of apoptosis in mast cells by different mechanisms since IL-3 can induce bcl-2 induction but SCF cannot [Yec, N. S., I. Peak and P. Besmer, 1994 role of the c-kit ligand in proliferation and suppression of apoptosis in mast cells. Basis for radiosensitivity of white spotting and steel mutants. J. Exp. Med. 179:1777-1787.]. Despite the wide-ranging ability of bcl-2 to promote cell survival, there are circumstances where bcl-2 is not found to be responsible for protecting the cells from apoptosis [Hueber, A., G. Raposo, M. Pierres and H. He. 1994. Thy-1 triggers mouse thymocyte apoptosis through a bcl-2-resistant mechanism. J Exp Med 179:785-96.]. Therefore, it is possible that other members of the bcl-2 family may provide protective effects in some specific biological processes. In monocytes IgE-receptor aggregation leads to up-regulation of bcl-XL and bcl-2 (N. Katoh, S. Kraft, J. H. M. Wessendorf, T. Bieber. The high-affinity IgE receptor (Fexcex5RI) blocks apoptosis in normal human monocytes. J. Clin. Invest. 105: 183-190 (2000)).
Al was originally identified from mouse bone marrow culture induced with granulocyte macrophage colony stimulating factor (GM-CSF) and bears structural homology to bcl-2 [Lin, E., A. Orlofsky, M. Berger and M. Prystowsky. 1993. Characterization of Al, a novel hemopoietic-specific early-response gene with sequence similarity to bcl-2. J Immunol 151:1979-88.]. Al was described as an early-response gene, expressed in multiple tissues such as thymus, spleen and bone marrow, and also expressed in a number of hemotopoietic cell lineages under various stimulations. These cells include T and B lymphocytes, macrophages, neutrophils [Chuang, P. I., E. Yee, A. Karsan, R. K. Winn and J. M. Harlan. 1998 Al is a constitutive and inducible Bcl-2 homologue in mature human neutrophils. Biochem. Biophys. Res. Commun, 249:361-5.], and endothelial cells [Lin, 1993, supra]. Al or the human homologue bfl-1 [S. S. Choi, S. H. Park, U. -J. Kim, H. -S. Shin. Bfl-1, a Bcl-2-related gene, is the human homolog of the murine Al and maps to Chromosome 15q24.3. Mammalian Genome 8, 781-782 (1997); Choi S. S., Park I. C., Yun J. W., Sung Y. C., Hong S. I., Shin H. S. A novel Bcl-2 related gene, Bfl-1 is overexpressed in stomach cancer and preferentially expressed in bone marrow. Oncogene Nov. 2, 1995; 11(9):1693-8] is the only bcl-2 family member that is inducible by inflammatory cytokines such as tumor necrosis factor-xcex1 and IL-1xcex2 suggesting a possible role in mast cell biology.
Available drugs for allergic diseases treat the symptoms of the disease which occurs as a consequence of released mediators from inflammatory cells. A treatment that more specifically would prevent the allergic response, with reduced side effects, compared to current drugs, would be desirable.
Accordingly an object of the present invention is to provide a method for treatment of mast cell mediated inflammation.
The object of the invention is obtained by a method as claimed in the claims. According to the invention a method of treatment of mast cell mediated inflammatory disorders in mammals is provided. The method comprises regulating the longevity and activity of activated mast cells by inhibiting the expression and/or function of the anti-apoptotic A-1/bfl-1 gene or gene product. In a preferred embodiment the inhibition of the expression and/or function of the A-1/bfl-1 gene or gene product is obtained by targeting inflamed tissue in the affected mammal with a drug that inhibits the function.
According to a further aspect of the invention there is provided use of a compound capable of inhibiting the expression and/or function of the anti-apoptotic A-1/bfl-1 gene or gene product for the manufacture of a medicament for treating mast cell mediated inflammatory disorders in mammals by regulating the longevity and activity of activated mast cells.
According to a further aspect of the invention, a biological or chemical assay for identifying compounds for treatment of mast cell mediated inflammatory disorders in mammals is provided. The assay comprises using the anti-apoptotic A-1/bfl-1 gene or gene product as a molecular target.
According to another aspect of the invention, a method for development of drugs for treatment of mast cell mediated inflammatory disorders in mammals is provided. The method comprises using the anti-apoptotic A-1/bfl-1 gene or gene product as a molecular target.
According to the invention it was surpringly found that by inhibiting the anti-apoptotic function of A-1/bfl-1 gene or gene product the activated mast cells undergo apoptosis, preventing re-granulation and re-activation. In that way the number of activated mast cells in the inflammatory tissue can be reduced and the allergic symptoms alleviated. The present invention provides a means to selectively target and eliminate mast cells that are actively involved in mediating the inflammatory response.
From U.S. Pat. No. 6,001,992 targeting pro-survival genes with antisense compounds is known. U.S. Pat. No. 5,843,773 discloses the sequences of bfl-1 and the relation to cancer. Mast cell mediated inflammations are not disclosed.
To investigate the role of Al/bfl-1 expression and function for the longevity of activated mast cells, a number of experiments were performed using mouse and human mast cells.
Mast cell cultures. Bone marrow-derived cultured mouse mast cells (BMCMC) were obtained by culturing mouse (C57BL/6 or Alxe2x88x92/xe2x88x92 [Hamasaki, A., F. Sendo, K. Nakayama, N. Ishida, I. Negishi, K. Nakayama and S. Hatakeyama. 1998. Accelerated neutrophil apoptosis in mice lacking Al-a, a subtype of the bcl-2-related Al gene. J Exp Med 188:1985-92.], 4- to 5-week old,) bone marrow cells for 5 to 6 weeks in 10% WEHI-3 (IL-3 producing cell-line) enriched conditioned RPMI 1640 medium, supplemented with 10% heat-inactivated fetal bovine serum (Gibco-BRL, Germany), 4 mM L-glutamine, 50 xcexcM 2-mercaptoethanol (Sigma Chemical Co., St. Louis, Mo.), 1 mM sodium pyruvate (Sigma), 0.1 mM non-essential amino acids (Sigma), 10 mM Hepes (Sigma), and 100 xcexcg/ml penicillin/streptomycin. The mast cell differentiation was confirmed by toluidine blue staining. The growth factor dependent mouse mast cell line MCP5/L was maintained in the same medium described above. For cell viability assays, the cell suspension was mixed with the vital dye, trypan blue, and only the number of live cells was scored.
Cord blood cultured human mast cells (CBCMC) were differentiated from cord blood cells cultured for 8-10 weeks in 50 ng/ml SCF and 10 ng/ml IL-6 (Pepro Tech, London UK) and complete RPMI medium. The purity of human mast cells was  greater than 95% by tryptase staining.
Activation of mast cells in vitro. For Fcxcex5RI-dependent activation, MCP5/L cells were resuspended at 1xc3x97106 cells/ml and sensitized using a monoclonal murine IgE anti-TNP (trinitrophenyl) (IgEl-b4, ATCC, Manassas, Va.) antibody (15% hybridoma supernatant) for 90 min. The cells were washed twice with warm medium, followed by challenge with 1 xcexcg/ml TNP-BSA for time periods indicated. For cell viability assays, both the anitbody sensitization and the anitgen challenge were performed in RPMI medium supplemented with 0.5% BSA (Sigma) at 37xc2x0 C. in a humidified incubator containing 5% CO2. For gene regulation studies, all the incubations were carried out in RPMI medium supplemented with 5% fetal bovine serum and antibiotics on a rocker platform. In some experiments, cells were cross-linked in the medium containing 1 mM EDTA or cells were incubated in ice-cold water during Fcxcex5RI cross-linking. Where indicated, various inhibitors were introduced to test their effects on A1 regulation after Fcxcex5RI cross-linking. These include bisindolylmaleimid (100 nM), wortmannin (100 nM), SB 203580 (pyridinylimidazole compound, inhibitor of p 38 mitogen-activated protein kinase) (1 xcexcM), PD 98059 (2-(2-amino-3-methoxyphenyl)-4-oxo-4H-[1]benzopyran, inhibitor of mitogen-activated protein kinase Kinase-1 (MEK1)) (10 xcexcM), genistein (1 xcexcM), suramin (100 xcexcM), cyclosporin A (2 xcexcg/ml), dexamethasone (1 xcexcM) and cycloheximide (10 xcexcg/ml). Dexamethasone and suramin were added 14 hr and 24 hr, respectively, before IgE sensitization. All the other inhibitors were added at the same time as Fcxcex5RI aggregation. In some A1 regulation assays, cells were resuspended in medium containing calcium ionophores (ionomycin) and compound 48/80 (N-methyl-p-methoxyphenethylamine) as well as cytokines including IL-3, IL-4, SCF, GM-CSF (granulocyte-macrophage colony stimulating factor) and TNF-xcex1 (tumour necrosis factor) and incubated for 6 hr at 37xc2x0 C. on a rocker platform. After various treatments mentioned above, cell pellets were collected and kept at xe2x88x9280xc2x0 C.
Human myeloma IgE (ND) and mouse anti-human IgE (clone 346) were used to activate CBCMC through cross-linkage of the high-affinity IgE-receptor. Human mast cells at 1xc3x97106 cells/ml were incubated with 1 xcexcg/ml IgE overnight, washed twice with PBS, plated at 3xc3x97105 cells/ml, and challenged with 2.0 xcexcg/ml anti-IgE for 30 min at 37xc2x0 C.
Cell viability measurement. Trypan blue exclusion assay was used in all of the cell survival experiments. In brief, cell suspension was mixed with the vital dye, trypan blue, and the number of live cells was scored. In some cases, cell apoptosis was also measured by Cell Death Detection ELISA (Boehringer Mannheim, Mannheim, Germany) quantitatively detecting the mono- and oligonucleosomes released into culture supernatant.
Isolation of RNA and RNAse Protection Assay (RPA) analysis. Total cellular RNA was isolated using the TriPure isolation reagent (Boehringer Mannheim, Mannheim, Germany). RPA was performed using the mAPO-2 and hAPO-2 multi-probe set from the RiboQuant System (PharMingen, San Diego, Calif.) following the supplier""s recommended protocol. Briefly, 10 xcexcg of RNA was hybridized overnight at 56xc2x0 C. with the 32P-labelled probes synthesized from the mAPO-2/h-APO-2 multi-probe template set. Protected fragments were precipitated and size fractionated by 5% polyacrylamide gels. Pixel intensity was determined using a phosphoimaging device. Levels of each gene transcript were quantified by MacBas V2.2 (Fuji Photo Film Co. Ltd., Japan).
Measurement of release. For detection of the granule-associated N-acetyl-xcex2-D-hexosaminidase (xcex2-hexosaminidase), an enzymatic colorimetric assay was used. Supernatant or a combination of supernatant and cell lysate was mixed with identical volume of substrate solution (7.5 mM p-nitrophenyl N-acetyl-xcex2-D-glucosaminide (Sigma) in 80 mM citric acid, pH 4.5), and incubated at 37xc2x0 C. for 2 hr. The reaction was stopped by adding glycine (0.2 M, pH 10.7) into each well and the absorbance was measured. Percentage of xcex2-hexosaminidase release is calculated as the absorbance of the supernatant over that of the combination of supernatant and lysate.
Statistics were calculated using an analysis of variance (ANOVA), followed by multiple comparison using Fisher""s method. * denotes p less than 0.05, ** p less than 0.01. Values presented are the meansxc2x1SEM.